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1. IF=7.725 Xingyun Wang

2. Backgrounds
1918
“Warburg effect”
Warburg discovered that thin slices of tumors produced lactate much more rapidly than normal tissue, even in the
presence of ample oxygen.
Warburg proposed that the rapid glucose fermentation and associated lactate secretion by the cancer cells was due to
mitochondrial dysfunction
Trends in Biochemical Sciences 2016 41, 211-218DOI: (10.1016/j.tibs.2015.12.001)

3. PTEN
2004
“Oncogene-driven glucose uptake”
Instead of causing mitochondrial dysfunction, it was found that the mutations that cause cancer also promote the
breakdown of glucose in a process called glycolysis.
The most striking example involves the PI3K-Akt signaling pathway, which both transduces the signal from the
hormone insulin to drive glucose uptake, and is one of the most frequently mutated pathways in cancer.
One way this pathway can be activated is by the loss of a tumor suppressing enzyme called PTEN (Nature
441:424–430.).
Backgrounds

4. 2012
Cancer cells hijack surrounding cells
Cancer cells secrete growth factors to promote the formation of new blood vessels (Orimo et al., 2005),
which are required to supply tumors with nutrients. (Cell 121:335–348.)
They co-opt surrounding connective tissue cells, including fibroblasts, which exchange signals with the
cancer cells in a manner that ultimately drives tumor growth and likely helps to suppress immune responses
to the tumor.(Cancer Metastasis Reviews 31:195–208.)
Fibroblasts may exchange both signaling molecules and metabolic fuels with the cancer cells, either by
secreting individual molecules (e.g. lactate;Seminars in Cancer Biology 25:47–60.) or by releasing
membrane-bound vesicles known as exosomes (Cancer Research 69:785–793.).
These exosomes contain small RNA molecules known as microRNAs that can silence the gene that
encodes the PTEN enzyme, whose loss drives an increase in glycolysis (Nature 527:100–104.)
Backgrounds

5. Cancer-associated fibroblasts release exosomes that both deliver nutrients to cancer cells and inhibit oxidative
phosphorylation
Backgrounds
Schematic shows the metabolic regulation of CDEs in cancer cells through inhibition of oxidative phosphorylation and
contribution of metabolite cargo. This regulation leads to significant increase of reductive glutamine metabolism in
cancer cells in presence of exosomes. CDEs are also cargo of amino acids, TCA cycle metabolites, and lipids. In
nutrient starved TME metabolites derived from exosomes enrich cancer cells with biosynthesis building blocks and
thereby promote tumor growth.

6. Backgrounds
1. The understanding of interaction mechanisms between cancer cells and the tumor microenvironment
(TME) is crucial for developing therapies that can arrest tumor progression and metastasis.
2. Although the TME is comprised of a variety of cell types including cancer-associated fibroblasts cells
(CAFs), immune cells, and angiogenic elements, CAFs are the major constituent of the TME in many cancers
3. Exosomes have emerged as a vital communication mechanism between different cell types in the TME.
4. Although it has been shown that CAFs can induce metabolic reprogramming in cancer cells (Brauer et al., 2013), the
contribution of CDEs in this phenomenon, if any, has not been elucidated
5. Most of the current studies are focused on cancer cell secreted exosomes; and little is known about CAF-derived
exosomes (CDEs) and their metabolic influence on cancer cells.
.

7. Results CDEs are internalized by prostate cancer cells
Exosomes isolated from conditioned media
obtained from patient-derived prostate CAFs
Flow analysis of Dynabeads conjugated with
anti-CD63 antibody
200 μg/ml
Flow cytometry analysis shows uptake of CAF
exosomes by prostate cancer cells.
Incubated with PKH67-labeled exosomes for 3

8. Results CDEs downregulate mitochondrial function of prostate cancer cells
CDEs enhanced proliferation
of PC3 cells with increasing
exosomes concentration
Basal oxidative phosphorylation
(OXPHOS, indicated by OCR) was
significantly inhibited with increasing
concentration of CDEs added to
PC3 cells
Cancer cell lines (PC3, DU145,
22RV1 and E006AA)

9. Results
Endocytosis inhibitor Cytochalasin
D (CytoD) 1.5 μg/ml
Maximal and reserve mitochondrial
capacities were measured using
FCCP and antimycin. Maximal OCR
is maximal capacity of mitochondrial
OCR. (n≥9)
Role of CAFs secreted exosomes
in regulating mitochondrial
membrane potential (MMP,膜电位)
of prostate cancer cells. MMP is an
important indicator of mitochondrial
functions.

10. Results
qPCR results show that mitochondrial OXPHOS genes of
prostate cancer cells were downregulated when cultured with
CDEs. (n=3).
Microarray
analysis
q-PCR
analysis
OXPHOS related ATP synthase complex
genes were downregulated in cells cultured
with CDEs.
Complexes IIIComplexes IV

11. Results
(miRNA-mRNA interactions taken from starBase v2.0, starbase.sysu.edu.cn)
Nanostring assays followed by miRNA target prediction integrated with
AGO-CLIP-SEQ data
The reduction of OCR is moderate,
this is due to the technical limitation of
co-transfection experiments which can
only allow using a small subset of the
miRNAs that target OXPHOS in PC3.
Co-transfected mir-22,
let7a and mir-125b

12. In summary, these results suggest that CDEs reduced mitochondrial
oxidative phosphorylation and induced metabolic alterations in
cancer cells mimicking hypoxia-induced alterations

13. Whether this reduced mitochondrial activity leads to increased glycolysis
in cancer cells in presence of CDEs?

14. ResultsCDEs upregulate glucose metabolism in cancer cells
These exosomes significantly increased glycolysis in
cancer cells when compared to cancer cells cultured
without exosomes.
CytoD partially inhibited this increase of ECAR, thus
confirming the role of exosomes in increase of glycolysis
in cancer cells .

15. Results
CDEs increased glucose uptake and lactate secretion when
compared to cancer cells cultured without exogenously added
exosomes.
Mass isotopologue distributions (MIDs):
M0 refers to the isotopologue with all 12C atoms and
M1 and higher refer to heavier isotopologues with one
or more 13C atoms derived from the tracer.

16. Results
乳酸 丙酮酸
The increase of M3 pyruvate and M3 lactate indicates higher contribution of glucose
to pyruvate and lactate in prostate cancer cells conditioned with CDEs.
Moreover, there was a decrease of M0 pyruvate and M0 lactate with a corresponding
increase of M1 pyruvate and M1 lactate, thus suggesting that the exosomes enhance
glycolysis.
The latter conclusion was based on the principle that M1 pyruvate is only produced
by glucose-6-phosphate metabolized by phospho glucoisomerase.

17. Results
柠檬酸盐 α-酮戊二酸 苹果酸盐
富马酸盐 谷氨酸 α-酮戊二酸
Consistent with the decreased OXPHOS observed in cancer cells due
to CDEs, the percentage of M2 citrate, M2 α-ketoglutarate, M2
fumarate, M2 malate, and M2 glutamate was also significantly
reduced in cancer cells in presence of CDEs
CDEs decreased the percentage
contribution of glucose to a-ketoglutarate
in cancer cells and instead diverted it
towards lactate

18. Results
The above results conclusively show that CDEs induce a Warburg
type phenotype in cancer cells, by disabling normal oxidative
mitochondrial function with a compensatory increase in glycolysis.
To further unravel the mechanistic links between disabled normal
oxidative mitochondrial function in cancer cells by CDEs and its
influence on cancer cells' mitochondrial metabolism.

19. Results CDEs enhance reductive pathway of glutamine metabolism in cancer cells
Using labeled U-13C5 glutamine.
谷氨酰胺 α-酮戊二酸
Proliferating cells under both normoxia and hypoxia can utilize glutamine by oxidative metabolism and produce
pyruvate through malic enzyme and further combine oxaloacetate with acetyl-CoA to form M4 citrate
Alternatively, proliferating cells under hypoxia have been reported to predominantly reductively carboxylate
glutamine generated α-ketoglutarate through IDH 1/2 to generate M5 citrate
CDEs increased M5 glutamate and M5 α-ketoglutarate in cancer cells thereby indicating that exosomes enhance
glutamine’s entry into TCA cycle

20. Results
柠檬酸 苹果酸 富马酸
Notably, there was significant increase in M5 citrate, M3 fumarate and M3 malate in cancer cells in
the presence of exogenously added exosomes thus suggesting that cancer cells rely critically on
reductive glutamine metabolism when normal mitochondrial function is disrupted by stromal
microenvironment

21. Results
To obtain mechanistic understanding of CDEs induced increased reductive carboxylation in cancer cells; we
measured the ratio of α-ketoglutarate to citrate abundance in cancer cells and found that exosomes increased this
ratio significantly
α-ketoglutarate to citrate was recently shown to promote reductive glutamine metabolism
The ratio of M4/M5 citrate, which represents the ratio of glutamine to citrate through oxidative metabolism over
reductive metabolism, confirmed our above results that there is a significant increase in glutamine’s reductive
metabolism in presence of exosomes

22. U-13C6 glucose or U-13C5 glutamine to estimate their conversion to cytosolic acetyl-CoA, which is the precursor
for palmitate (fatty acid) synthesis
Results
Lower mass isotopologues derived from U-13C6 glucose, when cells are cultured with exosomes.
Conversely, from low to high mass palmitate isotopologues derived from U-13C5 glutamine, in presence of CDEs
Isotopologue spectral analysis (ISA)indicated a significant decrease in the fraction of glucose contribution to lipogenic
acetyl-CoA in cancer cells cultured with exosomes.
More importantly, there is a two-fold increase in glutamine’s contribution to lipogenic acetyl-CoA via the reductive
carboxylation pathway.
Additionally, these intriguing results suggest that there are likely to be other sources apart from glucose and glutamine
that contribute to fatty acid synthesis.
Exosomes also decreased glucose contribution to palmitate.

23. Results
Acetate could be an important source for lipogenic acetyl-CoA in cancer cells, especially under hypoxic
conditions.
Interestingly, we noticed from the shift in palmitate mass isotopologues that CDEs increased acetate
contribution to lipids but decreased the pyruvate contribution.
U-13C3 pyruvate and U-13C2 acetate in cancer cells cultured with and without exosomes ( PC3 cells)

24. These experiments collectively substantiate that exosomes
have a significant effect on fatty acid synthesis in cancer cells
by switching the carbon source from the oxidative glucose
pathway to glutamine via the reductive carboxylation pathway
in the TCA cycle.

25. Results Intra-exosomal metabolomics reveal that CDEs contain an 'off-the-shelf' pool of metabolite cargo
High amounts of lactate and acetate in both prostate and
pancreatic CDEs
This suggests that exosomes can not only replenish TCA cycle
metabolites but also act as source of lipids.
TCA cycle metabolites, including pyruvate,
citrate, α-ketoglutarate, fumarate and
malate were measured using GC-MS in
exosomes isolated from pancreatic CAF35.

26. Results Ultra-high-performance liquid

27. Our results offer definitive proof for the first time that exosomes harbor
an 'off-the-shelf' pool of metabolite cargo, TCA cycle metabolites, amino
acids, and lipids, which can fuel the metabolic activity of the recipient
cells

28. Results CDEs can supply amino acids to cancer cells in a manner similar to macropinocytosis
To label metabolites, proteins and lipids in CAFs-secreted exosomes, CAFs were cultured in RPMI with labeled
13C3 pyruvate (pyr), 13C5 glutamine (gln), 13C6 leucine (leu), 13C6 lysine (lys), 13C9-phenylalanine (phe) and U-13C6
glucose.
Notably, our results substantiate that exosomes can supply metabolites to cancer cells under both complete and
nutrient deprivation conditions.

29. Results
To definitively prove the direct export of metabolites
by exosomes, we measured MIDs of metabolites in
cancer cells when cultured with 13C labeled
exosomes.
We detected substantial labeling of intracellular
amino acids in cancer cells, which included M5
glutamine, M6 lysine, and M6 leucine. We also
detected M5 glutamate derived from mitochondrial
glutaminolysis and labeled TCA cycle metabolites
from labeled 13C-glutamine supplied by CDEs

30. Results
CytoD, (1.5 μg/ml), heparin(50 μg/ml), and CQ
(chloroquine, 20 μM) inhibited this rescue of viability under
deprivation condition
EIPA: macropinocytosis inhibitor
CDEs were able to rescue reduction of proliferation under nutrient deprivation conditions (Figure 6C).
However, this rescue effect is reduced to varying extents by adding CytoD, heparin and lysosomal degradation inhibitor
choloroquine (Figure 6C).
Similarly, addition of macropinocytosis inhibitor EIPA also counters the rescue of CDEs under deprivation (Figure 6D).

31. These data suggest that uptake of exosomes and release of their
cargo is necessary to rescue cell proliferation under nutrient
deprived conditions.
Taken together, these results provide evidence that CDEs can
reprogram cancer cells’ metabolism by acting as source of amino
acids under nutrient depleted conditions in the TME.

32. Results CDEs supply metabolites to pancreatic cancer cells via Kras-independent mechanism
To expand the scope of our findings and understand whether Ras can similarly promote the
supply of metabolites by CDEs in pancreatic cancer cells, we isolated exosomes from pancreatic
CAFs cell line (CAF-19) and used them to study their metabolic influence in two pancreatic cancer
cell lines: BxPC3 (wild type Kras) and MiaPaCa-2 (homozygous Kras)
CDEs could rescue loss of
proliferation in both cancer cell lines,
thereby suggesting that
internalization or uptake and supply
of exosomes derived metabolites in
cancer cells is Kras independent
This rescue of proliferation by CDEs in pancreatic cancer cell
lines was inhibited by receptor mediated endocytosis inhibitor
heparin

33. Results
To further evaluate if exosomes mediated metabolic reprogramming is
Kras mediated, we measured mitochondrial respiration in PDAC cells with
and without pancreatic CDEs.
In line with results obtained in prostate cancer cells, we found that OCR of both BxPC3 and MiaPaCa-2 cells were
decreased in presence of pancreatic CDEs (Figure 7D).
Both maximal and reserve mitochondrial capacity of pancreatic cancer cells were significantly reduced in presence of
pancreatic CDEs further confirming mitochondrial respiratory capacity inhibition in cancer cells by CAF exosomes
(Figure 7D).
Furthermore, there was a corresponding increase in ECAR in both pancreatic cancer cell lines in presence of CDEs
(Figure 7E).
This was corroborated by increased lactate levels in the cancer cells in presence of exosomes using U-13C6 glucose
labeling based isotope tracer analysis of pancreatic cancer cells in presence of CAF exosomes (Figure 7F).

34. Concomitantly, CDEs decreased percentage contribution of glucose to α-
ketoglutarate in both pancreatic cancer cell lines
In line with the results obtained in prostate cancers, we found that exosomes from
pancreatic CAFs significantly increased the reductive glutamine metabolism
Results

35. Remarkably, this CAF exosomes-mediated increase of
reductive glutamine metabolism was detected in both wild-type
and activated Kras expressing pancreatic cancer cells, thus
suggesting that metabolic reprogramming induced by stromal
exosomes in cancer cells is not only Kras independent but is
broadly observed in many cancers.

36. Conclusions
Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid
cancers.
Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on
neoplastic cell-autonomous processes for these adaptations.
We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic
machinery following their uptake by cancer cells.
We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing
glycolysis and glutamine-dependent reductive carboxylation in cancer cells.
Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-
deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described
dependence on oncogenic-Kras signaling.
Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites,
including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central
carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.